Hybridoma cell line of secreting cyproheptadine monoclonal antibodies and preparation method thereof

a cyproheptadine monoclonal antibody and hybridoma cell technology, applied in the field of food safety and immunological detection, can solve the problems of large amount of drug residues in animals, poor manufacturers earned illegal profits, serious endanger human health, etc., and achieve good specificity and detection sensitivity

Active Publication Date: 2019-09-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0008]The purpose of the present invention is to obtain a kind of hybridoma cell line of secreting cyproheptadine monoclonal antibodies. The monoclonal antibody secreted by this hybrid tumor cell line has good specificity and detection sensitivity to cyproheptadine (IC50 value is 0.37 ng / mL), which could be used to establish the immunological detection method of cyproheptadine, and to detect the residues of cyproheptadine in food.

Problems solved by technology

As a result, many poor manufacturers earned illegal profits by adding the drug to animal feed and water to quickly gain weight.
However, this illegal behavior can lead to drug residues in animals, which accumulate in large quantities.
Since cyproheptadine can cause drowsiness, dizziness, weakness, nausea, overeating, weight gain, and even allergic shock, hemolytic anemia, mental disorder, ataxia, thick bronchial secretion, tachycardia, urinary retention and other adverse reactions, it seriously endangers human health.
However, these methods of pretreatment is complicated, time-consuming, and does not apply to a large number of samples of rapid detection.

Method used

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  • Hybridoma cell line of secreting cyproheptadine monoclonal antibodies and preparation method thereof
  • Hybridoma cell line of secreting cyproheptadine monoclonal antibodies and preparation method thereof
  • Hybridoma cell line of secreting cyproheptadine monoclonal antibodies and preparation method thereof

Examples

Experimental program
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Effect test

example 1

[0060]Synthesis of Cyproheptadine Hapten

[0061]5 g compound 1 of concentration of 17.4 mmol was dissolved in 50 mL methanol solution, and 3 g ethyl chloroformate of concentration of 34.8 mmol was added for stirring under 120° C. for the night to obtain mixture. the cooled mixture mentioned above was added HCL, extracted three times with 50 mL ethyl acetate, the extracted organic layer was dried with anhydrous Na2SO4, after vacuum concentration the 4 g compound 2 was obtained. 4 g compound 2 of concentration of 11.6 mmol was dissolved in 64 mL ethyl alcohol and added dropwise potassium hydroxide aqueous solution of equivalent of 15 to stir under 30° C. for the night to get mixture, which the aqueous solution of potassium hydroxide is 10.3 g potassium hydroxide dissolved in 16 mL H2O to form a solution with a concentration of 174 mmol. the mixture was extracted three times with 50 mL ethyl after acetatevacuum concentration, and the extracted organic layer was dried with anhydrous Na2SO...

example 2

[0062]Synthesis of Cyproheptadine Complete Antigen

[0063]5 mg cyproheptadine haptens was taken into brown flask adding 1 mL MES buffer solution of pH4.7 and the concentration of 0.1M to dissolve, 10 mg EDC and 6 mg NHS was added stirring for 6-8 h to get the activated liquid; the keyhole limpet haemocyanin KLH was dissolved in CB solution, the activated liquid was added adjusting pH to 9.0 with NaOH of the concentration of 1M, keeping in the dark and reacting for night. After isolating complete antigens and uncoupled small molecules, and identified by uv method, the cyproheptadine complete antigen was obtained (the uv characterization effect of cyheptadine complete antigen of the present invention is shown in FIG. 2).

example 3

[0064]Preparation of hybrid tumor cell lines that secrete the monoclonal antibody of cyproheptadine

[0065]Animal Immunization

[0066]After the cyproheptadine complete antigen and equivalent freund's adjuvant were mixed and emulsified, BALB / c mice were immunized by subcutaneous injection in the neck and back. For the first immunization, complete freund's adjuvant was used. For two times booster immunization, incomplete freund's adjuvant was used. The blood collection is performed on the seventh day after the end of the third immune process. Blood samples were taken from the mice after the above immune process, and the serum immune titer and immunosuppressive ability were detected by indirect ELISA to select the mice with high cyproheptadine antibody content in serum. The selected mice were subjected to 4th booster immunization with incomplete freund's adjuvant, and then, the rush immunization is performed via intraperitoneal injection, using cyproheptadine complete antigen without freun...

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Abstract

A hybridoma cell line of secreting cyproheptadine monoclonal antibodies with a preservation number of CGMCC No. 14699 belongs to the field of food safety immunological detection. BALB/c mice are immunized through one time immunization with complete freund's adjuvant, three times of booster immunization with incomplete freund's adjuvant and one time of rush immunization with cyproheptadine complete antigen without adjuvant; the spleen cells from BALB/C mice immunized with high potency and low value of IC50 are fused with murine myeloma cells; and then the hybridoma cell line is obtained through indirect competitive ELISA screening and three sub-clones. The monoclonal antibody secreted by this cell line has good specificity and detection sensitivity to cyproheptadine (value of IC50 is 0.37 ng/ml), being suitable for detection of cyproheptadine in food.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS[0001]This application claims priority from China Patent Application Serial Number 201810219839.2, which was filed on Mar. 16, 2018, the entire content of which is incorporated herein as reference.BACKGROUND OF THE INVENTION1. Technical Field[0002]The invention relates to the hybridoma cell line of secreting cyproheptadine monoclonal antibodies and preparation method thereof, belongs to the technical field of food safety and immunological detection.2. Background Art[0003]Cyproheptadine (CHP) is an antihistamine and an H1 receptor antagonist, and has the effect of anti-5-hydroxytryptamine, which can inhibit the satiety center of the hypothalamus to stimulate appetite, increase weight and promote growth. As a result, many poor manufacturers earned illegal profits by adding the drug to animal feed and water to quickly gain weight.[0004]However, this illegal behavior can lead to drug residues in animals, which accumulate in large quantities. When ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/44C07K16/12C07K16/06G01N33/12C12N5/09
CPCC12N5/0693C07K16/065G01N33/12C07K16/44C07K16/1289G01N33/577C07D211/70C07K14/795C07K19/00C07K2317/35G01N2430/00G01N33/9406C12N5/163
Inventor XU, CHUANLAIKUANG, HUAJIANG, WEIXU, LIGUANGMA, WEILIU, LIQIANGWU, XIAOLINGSONG, SHANSHANHU, YONGMING
Owner JIANGNAN UNIV
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